ABSTRACT Ikaros deficient large pre-B cells show attenuation of pre-BCR signaling and differentiation and an increase in stroma adhesion, and self-renewal. Ikaros plays a fundamentally unique role in the regulation of both active and cryptic Super-Enhancer networks (SE) in pre-B cells. Active SE supporting pre-B cell differentiation are defined by Ikaros and other B cell master regulators. However, they are uniquely dependent on Ikaros for their highly permissive chromatin environment and transcription activity. Ikaros, without any B cell transcription factors, is highly enriched at inactive enhancers of genes normally expressed in epithelial-stem cells. Upon Ikaros loss, expression of pre-B cell differentiation genes is attenuated, while a group of extra-lineage transcription factors, directly repressed by Ikaros, is induced. These collaborate with native B cell transcription regulators to induce a hybrid SE network that supports epithelial cell functions. This model of a dual mechanism of IKAROS regulation in promoting normal B cell differentiation while safeguarding against an epithelial-B cell phenotype and how deregulation contributes to B-ALL is investigated by three specific aims. Specific Aim 1. Regulation of pre-B cell super-enhancers and chromosomal domains. The role of Ikaros in the recruitment of chromatin modifiers and the mediator complex, key steps in SE activation, will be tested. We will also investigate whether Ikaros promotes the segregation of developmentally-affiliated genes into topological associated domains (TAD) domains. Specific Aim 2. Regulation of a cryptic transcriptional network in pre-B cells. Ikaros is engaged in the active repression of a regulatory network of transcription factors in WT pre-B cells that are normally prevalent in epithelial tissues. The contribution of these extra-lineage transcription factors to an epithelial-pre-B cell phenotype will be examined in the absence and presence of Ikaros. We will also investigate whether Ikaros provides an additional layer of protection by directly interfering with their activities at their enhancer target sites. Specific Aim 3. How a pre-leukemic transcriptional landscape is utilized for leukemia progression. Activating mutations in tyrosine kinases affiliated with growth factor receptor signaling support transformation of Ikaros mutant pre-B cells to a highly aggressive leukemic state. A key question addressed here is whether and how the leukemia-inducing signaling pathways utilize the epigenetic and transcription environment established by loss of Ikaros. Further studies on the potential conservation of these mechanisms in human B- ALL will be tested.